A Potent Chemotherapeutic Strategy In Bladder Cancer: S (MeO) TLC, A Novel Eg5 Inhibitor

BackgroundBladder urothelial carcinoma (UC) is the fifth most common malignancy worldwide, and the large majority (75%) present as non-muscle invasive tumorsl, which mainly confined to the mucosa (Ta) or subepithelial layer (T1). Despite received standard transurethral resection (TUR) and adjuvant intravesical chemotherapeutic instillations, about two-thirds of patients recur and 15% to 30% will progress to a higher stage and/or grade with greater mortality. Besides the clinical parameter (e.g. grade, stage), a number of markers were expected to predict the prognosis of non-muscle invasive UC, but few of them has been recognized as reliable method till now. Thus, there is a considerable need for investigating effective markers that can accurately identify non-muscle invasive cases with a high risk of recurrence or progression, which could contribute to not only the improvement of the prognosis by treating such individual patients more aggressively but also development of novel anticancer target.Invasive bladder cancer, which accounts for 25% of all bladder cancers, has a poor prognosis with low 5-year survival levels of 10～50%. Traditionally, the combination of methotrexate, vinblastine, doxorubicin and cisplatin (MVAC) has been adopted for such advanced cases with systemic spreading of disease. However, these chemotherapy modalities only deliver a 46～60% response rate. Although new combination of gemcitabine and cisplatin has been identified as first line chemotherapy for less toxicity, no better response rate was found. To overcome the limited efficacy of these conventional chemotherapies for advanced bladder cancer, novel therapeutic approaches are required.The mitotic kinesin Eg5, a member of the kinesin-5 family which plays a crucial role in the formation and maintenance of the bipolar spindle during mitosis, has been identified as an attractive target for cancer therapies. Since the discovery of the first Eg5 inhibitor, monastrol, many Eg5 inhibitors have been demonstrated to have anticancer efficacy.In consistent with the fact that Eg5 is only expressed in mitotic cells, especially in malignant cells, Eg5 expression levels appeared to be directly proportional to the mitotic population in both cancer cell lines and clinical tumors. Furthermore, Eg5 has been shown to correlate with oncogenesis, proliferative rate and clinical outcomes in cancers. However, to our knowledge, there was no data on the correlation between Eg5 expression and the prognosis of UC.Among the recognized Eg5 inhibitors, DeBonis et al. first identified (S)-trityl-L-cystein (STLC) as an effective Eg5 inhibitor. This inhibitor showed antiproliferative activity that was 36 times more potent than monastrol. Recently, it has been found by ourselves and other investigators that S(MeO)TLC, a (S)-trytyl-L-cystein derivative, is 10-fold more potent than STLC in cytotoxic activity. Furthermore, we also identified KPYC10665, KPYC10666 and KPYC10728, which are functionalized carbazoles prepared by one-pot N-arylation-oxidative biaryl coupling of anilines and phenyl triflates. Although there have been no reports concerning bladder tumors, the expression of Eg5 have been found to be induced in several kinds of tumors, including malignant melanoma, lung cancers and squamous cell carcinoma of the head and neck.In the present study we investigated the potency and specificity of several Eg5 inhibitors of bladder cancer cell proliferation. Furthermore, the antitumor effect of the most prominent inhibitor was examined in xenograft models to assess whether it could have potential in a clinical setting. Besides, we analyzed the expression of Eg5 in clinical bladder UC specimens by immunohistochemistry, and evaluated the correlation between Eg5 expression and clinicopathological characteristics, aiming at identifying the evidence for its usefulness as a prognostic marker in patients with non-muscle invasive UCs.ObjectiveWe investigate the relationship between Eg5 expression and prognosis of patients with bladder cancer, and then examine the anticancer activity of a novel Eg5 inhibitor for bladder cancer, with particular reference to metastatic diseases.MethodsEg5 expression was examined by immunohistochemistry in bladder cancer specimens (Grade:Grade 1,32 cases; Grade 2,98 cases and Grade 3,63 cases. Stage: pTa 49 cases; pTl,114 cases; pT2-T4,30 cases), and the correlation between clinicopathological characteristics and Eg5 expression was evaluated. The prognostic significance of Eg5 immunoreactivity was analyzed via survival analysis in 163 non-muscle invasive bladder cases that were treated with transurethral resection and adjuvant intravesical instillations.Bladder cancer cell lines were also examined for Eg5 expression, including RT112, KU7, RT4, EJ cell lines etc. The anti-proliferative activity of 5 Eg5 inhibitors was analyzed in cell lines using cell viability assay. The anticancer efficacy of the most potent Eg5 inhibitor was investigated in vitro using an apoptosis assay with Hoechst nuclear staining and flow cytometry. Immunofluorescence and immunostaining were used to elucidate the inhibitory mechanism. Furthermore, the inhibitory effect was evaluated in vivo using subcutaneous xenografts and metastatic cancer models.ResultsThe expression of Eg5 was significantly associated with tumor grade (P= 0.002) and stage (P= 0.021).163 patients with non-muscle invasive bladder cancer were regularly followed up with the mean of 32.52 (from 6 to 72) months. Univariate analysis revealed Eg5 overexpression exhibited unfavorable influence on the intravesical recurrence with a significance (P= 0.012) while a marginal statistical significance on progression (P= 0.070), and the Eg5 overexpression had significant adverse impact on the progression free survival in T1 tumors (P= 0.041). Subsequent Cox hazard multivariate analysis revealed that both grade (P= 0.045) and Eg5 expression(P= 0.029) were identified as independent predictors for intravesical recurrence with adverse significance.Eg5 expressed in bladder cancer cell lines. (S)-methoxy-trityl-L-cystein, S(MeO)TLC, exhibited the strongest antiproliferative activity of the 5 Eg5 inhibitors, and induced cell death after mitotic arrest via the Caspase-dependent apoptotic pathway. In vivo, S(MeO)TLC effectively suppressed tumor growth in both subcutaneous and metastatic xenograft models. Moreover, the survival times of S(MeO)TLC-treated nude mice with metastases were significantly longer than those of untreated mice (P< 0.001).ConclusionEg5 overexpression correlates with poor differentiation and progression of bladder cancer, and has an independent validity in predicting the intravesical recurrence in patients with non-muscle invasive cases. We conclude that S(MeO)TLC is a promising novel anticancer agent for the treatment of bladder cancer. Importantly, our data indicates the potential of S(MeO)TLC as an effective therapy for metastatic bladder cancers. Additionally, this study was the first to use IVIS in investigating efficacy of novel chemotherapy in vivo metastatic models.